While the central melanocortin system is recognized as a key regulator of energy balance and appetite, there remains much to learn regarding non-melanocortin components of this pathway. The hypothalamic melanocortin receptor, MC4R, is a G-protein coupled receptor that is antagonized by the peptide ligand, agouti-related protein (AgRP), which leads to increased feeding and weight gain. Syndecan-3, a heparan sulfate proteoglycan, has previously been implicated in potentiating AgRP antagonism. This proposal aims to investigate the role of syndecans, both at a molecular level and in vivo. We hypothesize that AgRP binds the glycosaminoglycan (GAG) components of syndecans, and that this interaction increases the local concentration of the peptide near MC4R. Furthermore, we have previously shown that designed positive charge mutations to AgRP lead to increased in vivo efficacy that is independent of MC4R binding, and we hypothesize that this is due to an increased affinity for the negatively charged GAGs proximal to the receptor. This proposal will reveal the molecular details of the interaction between AgRP peptides and GAGs using calorimetry and magnetic resonance. Our preliminary calorimetry data show tight binding between AgRP and heparin, and this affinity is strengthened by additional peptide positive charge. We will also investigate the mechanism of syndecan modulation of AgRP using a syndecan knockout mouse line. Our charged peptides will be used to test whether these mice respond differently from their wild type littermates in terms of feeding and weight gain. This knockout line will also allow us to probe the local concentration effects of syndecans using immunohistochemical staining. Finally, we will continue to engineer AgRP mutants with increased positive charge in order to develop an even more potent peptide, and use head-to-tail cyclization to increase peptide stability. This will provide a model of a specific, bi-functional ligand that increases feeding. We expect the proposed research to contribute a definitive role for syndecans in the AgRP-MC4R pathway that leads to hyperphagia, weight gain, and prolonged feeding when stimulated. This contribution will be significant because the complexity of GPCR systems allows for exciting new therapeutic avenues to be specifically targeted as we increase our understanding of these pathways at a structural level